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Biology of Reproduction 64, 473-481 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Influence of the Degree of Stimulation of the Pituitary by Gonadotropin-Releasing Hormone on the Action of Inhibin and Testosterone to Suppress the Secretion of the Gonadotropins in Rams1

A.J. Tilbrook2,a, D.M. de Kretserb, and I.J. Clarkec

a Department of Physiology and b Institute of Reproduction and Development, Monash University, Victoria, 3800, Australia c Prince Henry's Institute of Medical Research, Clayton, Victoria, 3168, Australia

ABSTRACT

This experiment determined if the degree of stimulation of the pituitary gland by GnRH affects the suppressive actions of inhibin and testosterone on gonadotropin secretion in rams. Two groups (n = 5) of castrated adult rams underwent hypothalamopituitary disconnection and were given two i.v. injections of vehicle or 0.64 µg/kg of recombinant human inhibin A (rh-inhibin) 6 h apart when treated with i.m. injections of oil and testosterone propionate every 12 h for at least 7 days. Each treatment was administered when the rams were infused i.v. with 125 ng of GnRH every 4 h (i.e., slow-pulse frequency) and 125 ng of GnRH every hour (i.e., fast-pulse frequency). The FSH concentrations and LH pulse amplitude were lower and the LH concentrations higher during the fast GnRH pulse frequency. The GnRH pulse frequency did not influence the ability of rh-inhibin and testosterone to suppress FSH secretion. Testosterone did not affect LH secretion. Following rh-inhibin treatment, LH pulse amplitude decreased at the slow, but not at the fast, GnRH pulse frequency, and LH concentrations decreased at both GnRH pulse frequencies. We conclude that the degree of stimulation of the pituitary by GnRH does not influence the ability of inhibin or testosterone to suppress FSH secretion in rams. Inhibin may be capable of suppressing LH secretion under conditions of low GnRH.

anterior pituitary, FSH, GnRH, hormone action, inhibin, LH,, testosterone

INTRODUCTION

In rams, the testicular hormone inhibin plays a major role in the negative feedback regulation of the secretion of FSH through actions directly on the pituitary gland [1–4]. Testosterone is also able to act directly, and to synergize with inhibin, at the pituitary to inhibit the secretion of FSH [1], although these actions are influenced by the stage of the breeding season [3]. The pituitary actions of inhibin and testosterone were demonstrated in castrated rams that had undergone hypothalamopituitary disconnection (HPD) and been infused i.v. with GnRH every 2 h while treated with combinations of recombinant human inhibin A (rh-inhibin) and testosterone [1, 3]. We suggested that the actions of inhibin at the pituitary to suppress FSH secretion in rams may be influenced by the type of stimulation of the pituitary by GnRH [1]. Based on observations between different experiments, there appeared to be differences between HPD castrated rams receiving a fixed dose of GnRH and castrated rams with an intact hypothalamopituitary unit in the extent to which the plasma concentrations of FSH were suppressed following treatment with rh-inhibin [1]. The frequency of GnRH pulses acting on the pituitary in castrated rams with an intact hypothalamopituitary unit would have been greater than a pulse every 2 h [5–7], which was the frequency received by the HPD rams [1]. Following an i.v. injection of rh-inhibin, plasma concentrations of FSH were reduced by 31% in HPD castrated rams and by 20% in castrated rams with an intact hypothalamopituitary unit, implying that reduction or limitation of the GnRH drive to the pituitary may increase the effectiveness of the suppressive actions of inhibin on FSH secretion [1]. In contrast, we recently observed that the degree of suppression in FSH secretion following two i.v. injections of rh-inhibin appeared to be similar in HPD castrated rams receiving GnRH every 2 h and in castrated rams with an intact hypothalamopituitary unit [3]. Nevertheless, these comparisons were made between separate experiments, and they were not compared statistically. It remains to be determined in rams whether the pituitary actions of testicular hormones to regulate the secretion of FSH are influenced by the degree of stimulation of the pituitary by GnRH. This may be important, because stimulation of the pituitary by GnRH varies between different physiological conditions, such as between stages of the breeding season [8], levels of nutrition [9], sexual stimulation [10], and stress [11].

Whether the stimulation of the pituitary gland by GnRH influences the feedback actions of testicular hormones in rams is unknown, but evidence from other experimental paradigms suggests that the GnRH drive to the pituitary can affect the actions of inhibin. In particular, it has been suggested that the actions of inhibin to suppress FSH secretion in female rats are increased when stimulation of the pituitary by GnRH is reduced [12]. This conclusion was based on the finding that the greatest suppression of FSH secretion occurred when rh-inhibin was administered to rats in which GnRH secretion had already been abolished by either a GnRH antagonist or pharmacological doses of estradiol benzoate [12]. In contrast, treatment of ewes with a GnRH agonist did not affect the level of suppression in FSH secretion from pituitary cells in vitro due to treatment with inhibin or estradiol [13].

It seems to be unlikely that stimulation of the pituitary by GnRH influences the negative feedback regulation of LH secretion in rams. Administration of rh-inhibin to rams has not affected the secretion of LH [2–4], and testosterone has negligible actions at the pituitary to affect the secretion of LH [3, 7]. To improve our understanding of the interactions between GnRH, inhibin, and testosterone at the pituitary in males, this experiment aimed to determine if the feedback effects of inhibin and testosterone at the pituitary to affect the secretion of gonadotropins in rams are influenced by the degree of pituitary stimulation by GnRH. We used HPD castrated rams treated with rh-inhibin and testosterone, both alone and in combination, at two frequencies of GnRH pulses to test two hypotheses: that the pituitary actions of inhibin and testosterone to suppress the secretion of FSH are increased when the frequency of GnRH pulses is decreased, and that inhibin and testosterone do not act at the pituitary to affect the secretion of LH irrespective of the degree of pituitary stimulation by GnRH.

MATERIALS AND METHODS

Animals

This experiment used ten 3-yr-old, adult Romney Marsh rams that had been castrated within the first 3 wk of birth and was conducted at the Victorian Institute of Animal Science, Werribee, Australia (38°S latitude) during the breeding season for this breed of sheep [14]. These castrated rams are similar to those we used in previous studies to investigate the feedback effects of testicular hormones [1–4, 7]. The mean (± SEM) live weight of the castrated rams was 41.9 ± 1.5 kg. For the duration of the experiment, the animals were penned individually in an animal house and offered a maintenance ration and water ad libitum.

Two weeks before the experiment, the castrated rams underwent HPD as described by Clarke et al. [15]. From the time of HPD and throughout the experimental period, each castrated ram received a pulsatile i.v. infusion of 125 ng of GnRH per pulse (Auspep, South Melbourne, Australia) using an automatically programmed pump as previously validated by us [7]. The frequency of GnRH pulses was varied depending on the stage of the experiment (see below). Each animal was fitted with an indwelling catheter (Dwellcath; Tuta Laboratories, Lane Cove, Australia) in each jugular vein. One catheter was used to infuse GnRH and the other to collect blood samples and to inject vehicle or rh-inhibin.

The care and use of the animals in this experiment conformed with the requirements of the Australian Prevention of Cruelty to Animals Act of 1986 and with the National Health and Medical Research Council/CSIRO/AAC code of practice for the care and use of animals for scientific purposes.

Experimental Procedure

The castrated rams were allocated to two groups of five each and were treated i.v. with vehicle or rh-inhibin and i.m. with an oil vehicle or testosterone propionate while being administered GnRH at each of two pulse frequencies (i.e., fast and slow) (Fig. 1).



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  		FIG. 1. Design of the experiment. Two groups of castrated rams (n = 5/group) received either two i.v. injections of vehicle (group 1) or rh-inhibin (group 2, 0.64 µg/kg). All animals were treated with i.m. injections of oil every 12 h when infused with 125 ng of GnRH every 4 h (slow GnRH pulse frequency) for 4 days and 125 ng of GnRH every hour (fast GnRH pulse frequency) for 4 days. The procedure was then repeated when the animals were treated i.m. with 16 mg of testosterone propionate every 12 h. For the first 3 days of treatment with oil, for 3 days between receiving the slow and fast GnRH pulse frequencies, and for the first 3 days of treatment with testosterone propionate, the castrated rams received 125 ng of GnRH every 2 h. On Days 1, 7, 14, and 21 (asterisk), blood samples were taken over 8 h before and after two injections with vehicle (group 1) or rh-inhibin (group 2). The first injection of vehicle (group 1) or rh-inhibin (group 2) was given 6 h after the end of the first sampling period, and the second injection was given 6 h after the first and 4 h before commencement of the second sampling period

The vehicle or rh-inhibin (0.64 µg/kg) [1, 3, 4] was given as two i.v. injections 6 h apart [3]. Blood samples were collected over 8 h before and after treatment. The first injection of vehicle or rh-inhibin was given 6 h after the end of the first period of sampling, and the second injection was given 6 h later (4 h before the second period of sampling). During each period of sampling, blood samples were collected at -10, 10, 20, 30, 40, and 60 min relative to the start of each GnRH pulse (Fig. 1). The plasma was assayed for FSH, LH, and testosterone.

During the periods of blood sampling and treatment with vehicle or rh-inhibin, GnRH was administered as one pulse every 4 h (i.e., slow-pulse frequency) or one pulse per hour (i.e., fast-pulse frequency). These frequencies of GnRH pulses were imposed for 4 days before the sampling and treatment periods. Between these times, the castrated rams received a pulse of GnRH every 2 h for 3 days (Fig. 1). Oil and testosterone propionate were given as i.m. injections every 12 h for at least 7 days, as described by Tilbrook et al. [7]. The dose of testosterone propionate used was 16 mg every 12 h, which yields parameters of plasma LH and testosterone similar to those of intact rams [7].

Preparation of rh-Inhibin and Vehicle

The rh-inhibin and vehicle used in this experiment were identical to those reported previously by us [1–4]. Briefly, the rh-inhibin had been purified from a recombinant mammalian cell line [16] and was obtained from Biotech Australia Pty Ltd. (Sydney, Australia). The physicochemical and biological characteristics of this rh-inhibin are similar in almost all respects to those of inhibin isolated from other species. The rh-inhibin preparation consisted of a mixture of two molecular weight forms (31 and 34 kDa) of inhibin of similar bioactivities in vitro, with the proportion of 31-kDa rh-inhibin being between 80% and 90% [17]. The rh-inhibin was originally stored in approximately 35% acetonitrile/0.1% trifluroacetic acid at -70°C, and each sample was thawed, BSA (0.1% final concentration) added, the acetonitrile removed by evaporation under nitrogen, and the remaining sample gel-filtered (Sephadex G-25, PD 10 columns; Pharmacia, Uppsala, Sweden) in Dulbecco's phosphate buffer (pH 7.2). Aliquots of the inhibin fraction were stored at -70°C. For use in the experiment, the rh-inhibin sample was diluted in sterile, 0.9% NaCl to the appropriate concentration. The vehicle consisted of appropriate volumes of the phosphate buffer and saline identical to the volumes of rh-inhibin and saline used in each case.

Radioimmunoassays

Plasma concentrations of FSH were measured by radioimmunoassay as described by Bremner et al. [18] using NIADDK-oFSH-RP1 as the standard. Five assays were conducted with a mean (± SEM) assay sensitivity of 0.17 ± 0.03 ng/ml, a range in sensitivities of 0.1–0.3 ng/ml, and a maximum point of precision of 4.1%–7.8% at 4.7–5.6 ng/ml. The intraassay coefficient of variation of less than 10% was between 0.7–1.9 and 11.0–23.0 ng/ml, and the interassay coefficient of variation was 3.8% at 2.4 ng/ml and 6.1% at 11.8 ng/ml.

The radioimmunoassays used to measure plasma concentrations of LH were conducted as described by Lee [19] using National Institute of Health LH S18 as the standard. Five assays were conducted with a mean (± SEM) assay sensitivity of 0.20 ± 0.03 ng/ml, a range in sensitivities of 0.2–0.3 ng/ml, and a maximum point of precision of 0.9%–0.10% at 4.6–7.3 ng/ml. The intraassay coefficient of variation of less than 10% was between 1.5–6.2 and 8.3–17.0 ng/ml, and the interassay coefficient of variation was 10.9% at 1.4 ng/ml and 11.8% at 2.5 ng/ml.

Two samples from each castrated ram were assayed for testosterone. The method described by Young et al. [20] was used after ethyl acetate-hexane extraction. All samples were measured in one assay with a sensitivity of 0.1 ng/ml and intraassay coefficient of variation of 7.9% at 3.2 ng/ml and 11.2% at 5.0 ng/ml. The plasma concentrations of testosterone were less than the sensitivity of the assay in castrated rams treated with oil, whereas the mean (± SEM) plasma concentration of testosterone in castrated rams treated with testosterone propionate was 8.2 ± 5.7 ng/ml.

Statistical Analyses

Repeated-measures analysis of variance was used to analyze the effects of treatments on the plasma concentrations of FSH and LH and the amplitude of LH pulses. The means of the plasma concentrations (ng/ml) of FSH and LH for the sampling period before (i.e., pretreatment) and following (i.e., posttreatment) the injections of vehicle or rh-inhibin were compared. For LH, the means of the amplitude of the pulses during the pretreatment and posttreatment sampling periods were also compared. The amplitude of LH pulses (ng/ml) were defined as the difference between the maximal concentration of LH that occurred following the delivery of a GnRH pulse and the concentration of the sample immediately before commencement of the GnRH pulse. The between-subjects factor was treatment with vehicle or rh-inhibin, and the repeat variables were steroid (i.e., oil and testosterone propionate), GnRH pulse frequency (i.e., slow and fast), and period (i.e., pretreatment and posttreatment). All data were checked for homogeneity of variance, and transformation of data was not necessary. When appropriate, paired comparisons were made using least significant differences. In Figures 2, 3, and 4, overall effects of main factors are presented, because no significant interactions were observed between factors.



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  		FIG. 2. Overall mean (± SEM) plasma concentrations of FSH (ng/ml), plasma concentrations of LH (ng/ml), and amplitude of LH pulses (ng/ml) in the HPD castrated rams when receiving one pulse of GnRH every 4 h (slow) and one pulse of GnRH every hour (fast). No significant interactions between factors were observed. The asterisk indicates a significant (P < 0.01) difference between pulse frequencies

RESULTS

Follicle-Stimulating Hormone

Overall, the mean (± SEM) plasma concentrations of FSH (Fig. 2) were significantly (P < 0.05) greater when the castrated rams were receiving the slow (one pulse every 4 h) GnRH pulse frequency (4.8 ± 0.3 ng/ml) than when receiving the fast (one pulse every hour) GnRH pulse frequency (4.1 ± 0.4 ng/ml). No significant interactions involving treatment with vehicle or rh-inhibin or treatment with oil and testosterone propionate were observed.

Treatment with oil or vehicle did not affect the plasma concentrations of FSH at either GnRH pulse frequency (Table 1). In contrast, FSH concentrations were significantly (P < 0.01) reduced by 30% following treatment with rh-inhibin (Fig. 3). No interaction was observed between treatment with vehicle or rh-inhibin and frequency of GnRH pulses, indicating that the frequency of GnRH pulses did not influence the suppressive effects of rh-inhibin on FSH secretion. Moreover, the suppressive effects of rh-inhibin on plasma concentrations of FSH were not influenced by treatment of the castrated rams with testosterone propionate (Table 1).


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  		TABLE 1. Mean (± SEM) plasma concentrations of FSH (ng/ml) in castrated rams treated with two i.v. injections of vehicle (group 1) or rh-inhibin (0.64 {µ}g/kg, group 2) and i.m. injections every 12 h of oil or 16 mg of testosterone propionate (TP) when administered 125 ng of GnRH every 4 h (slow pulse frequency) and 125 ng of GnRH every hour (fast pulse frequency) for 4 days.a



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  		FIG. 3. Overall mean (± SEM) plasma concentrations of FSH (ng/ml) of HPD castrated rams before (pretreatment) and after (posttreatment) treatment with vehicle (group 1) or rh-inhibin (group 2). No significant interactions between factors were observed. The asterisk indicates a significant (P < 0.01) difference between pre- and posttreatment in the castrated rams treated with rh-inhibin

Overall, the mean (± SEM) plasma concentrations of FSH in the HPD castrated rams were significantly (P < 0.01) lower during treatment with testosterone propionate (4.1 ± 0.4 ng/ml) than during treatment with oil (5.6 ± 0.5 ng/ml, 28.6% suppression). At the slow GnRH pulse frequency, the plasma FSH concentrations were 6.0 ± 0.6 ng/ml during treatment with oil and 4.6 ± 0.6 ng/ml during treatment with testosterone propionate (23.3% suppression), whereas at the fast GnRH pulse frequency, the FSH concentrations were 5.3 ± 0.8 and 3.6 ± 0.6 ng/ml during treatment with oil and testosterone propionate, respectively (32.1% suppression). Nevertheless, no interaction was observed between steroid treatment (i.e., oil or testosterone propionate) and GnRH pulse frequency, indicating that the suppression of FSH by testosterone propionate was not influenced by the frequency of GnRH pulses.

Luteinizing Hormone

Plasma concentrations Overall, the mean (± SEM) plasma concentrations of LH (Fig. 2) were significantly (P < 0.05) higher when the castrated rams were infused with the fast (one pulse every hour) GnRH pulse frequency (3.7 ± 0.2 ng/ml) than when they were infused with the slow (one pulse every 4 h) GnRH pulse frequency (2.9 ± 0.1 ng/ml). This difference was not influenced by treatment with vehicle or rh-inhibin or oil and testosterone propionate. No effect of treatment with vehicle (group 1) was observed on the mean plasma concentrations of LH (Table 2), but in castrated rams treated with rh-inhibin (group 2), a significant (P < 0.05) overall decrease was observed in mean plasma concentrations, from 3.8 ± 0.3 before to 2.6 ± 0.2 ng/ml after the injections of rh-inhibin. This occurred irrespective of the GnRH pulse frequency and treatment with oil or testosterone propionate (Table 2). No direct effect of treatment with testosterone propionate was observed on the plasma concentrations of LH.


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  		TABLE 2. Mean (± SEM) plasma concentrations of LH (ng/ml) in castrated rams treated with two i.v. injections of vehicle (group 1) or rh-inhibin (0.64 {µ}g/kg, group 2) and i.m. injections every 12 h of oil or 16 mg of testosterone propionate (TP) when administered 125 ng of GnRH every 4 h (slow pulse frequency) and 125 ng of GnRH every hour (fast pulse frequency) for 4 days.a

Pulse amplitude The overall mean (± SEM) amplitude of LH pulses (Fig. 2) during treatment of castrated rams with one pulse of GnRH every 4 h (4.8 ± 0.2 ng/ml) was significantly (P < 0.01) greater than during treatment with one pulse of GnRH every hour (2.6 ± 0.2 ng/ml). No interactions with other factors were observed.

The amplitude of LH pulses was not affected by treatment with oil or testosterone propionate, and it did not change significantly following injections of vehicle (group 1) (Table 3). In contrast, a significant (P < 0.05) decrease was observed in the mean amplitude of LH pulses following treatment with rh-inhibin (group 2) during the slow (one pulse every 4 h), but not during the fast (one pulse every hour), GnRH pulse frequency (Fig. 4). No interactions were observed between treatment with oil or testosterone propionate, treatment with vehicle or rh-inhibin, and frequency of GnRH pulses, indicating that the reduction in amplitude of LH pulses following treatment with rh-inhibin at the slow GnRH pulse frequency was not influenced by whether the castrated rams were treated with oil or testosterone propionate.


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  		TABLE 3. Mean (± SEM) amplitude of LH pulses (ng/ml) in castrated rams treated with two i.v. injections of vehicle (group 1) or rh-inhibin (0.64 {µ}g/kg, group 2) and i.m. injections every 12 h of oil or 16 mg of testosterone propionate (TP) when administered 125 ng of GnRH every 4 h (slow pulse frequency) and 125 ng of GnRH every hour (fast pulse frequency) for 4 days.a



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  		FIG. 4. Overall mean (± SEM) amplitude of LH pulses (ng/ml) in HPD castrated rams before (pretreatment) and after (posttreatment) treatment with vehicle (group 1) and rh-inhibin (group 2) when receiving one pulse of GnRH every 4 h (slow) and one pulse of GnRH every hour (fast). The asterisk indicates a significant (P < 0.01) difference between pre- and posttreatment for the rh-inhibin treated rams

DISCUSSION

These results clearly show that the level of stimulation of the pituitary gland by GnRH does not influence the actions of inhibin and testosterone to suppress the secretion of FSH or the actions of testosterone to affect the secretion of LH in rams. On the other hand, inhibin appears to be capable of acting on the pituitary of rams to reduce the secretion of LH when the level of pituitary stimulation by GnRH is low, because the amplitude of LH pulses was suppressed at the slow, but not at the fast, GnRH pulse frequency. Our findings differ from those of Rivier and Vale [12], who found that rh-inhibin was more effective at suppressing the secretion of FSH in female rats when the GnRH drive to the pituitary was reduced. Nonetheless, these studies used different experimental designs and were conducted with different species and sexes, which may explain the contrasting findings. Other studies, in which the findings have implied that GnRH may affect the pituitary actions of inhibin, also varied from ours, but those studies used follicular-fluid preparations rather than purified inhibin and were conducted with females [21, 22] or were in vitro studies using pituitary cell cultures from male rats [23]. Our results support findings in ewes, in which the actions of inhibin were not influenced by reducing the degree of pituitary stimulation by GnRH [13, 24]. The results of this experiment also differ from our observations in one study [1] but agree with those in another [3] comparing the suppressive effects of rh-inhibin on FSH secretion in HPD castrated rams and in castrated rams with an intact hypothalamopituitary unit. The current experiment was specifically designed to determine if the level of stimulation of the pituitary gland by GnRH affects the pituitary actions of inhibin and testosterone in rams. Clearly, the mechanisms by which inhibin and testosterone act directly at the pituitary gland to regulate the secretion of FSH in rams are not influenced by the frequency of GnRH pulses and, therefore, are unlikely to depend on events that are mediated via GnRH receptors.

As expected, treatment with rh-inhibin suppressed the plasma concentrations of FSH in the HPD castrated rams, confirming that inhibin negatively regulates the secretion of FSH [2] by actions directly at the pituitary gland [1, 3] in rams. This study also demonstrated that testosterone can act directly at the pituitary gland of rams to suppress the secretion of FSH during the breeding season, because the plasma concentrations of FSH were suppressed when the HPD castrated rams were treated with testosterone propionate. The extent to which testosterone suppressed the secretion of FSH in the HPD castrated rams in this experiment is similar to our recent findings for the actions of testosterone on FSH secretion in rams during the breeding season [3]. We found that during the nonbreeding season, but not during the breeding season, inhibin and testosterone synergized at the pituitary to negatively regulate the secretion of FSH in rams [3]. The current study was conducted during the breeding season, and as anticipated, no synergy was observed between the actions of inhibin and testosterone to suppress the secretion of FSH. Although both inhibin and testosterone suppressed the secretion of FSH, the plasma concentrations were suppressed to a greater extent following treatment with rh-inhibin than with testosterone propionate, and maximal suppression occurred when the HPD castrated rams were treated with both testicular hormones. These findings support our previous conclusions that inhibin is more potent than testosterone as a negative feedback regulator of the secretion of FSH in rams [1–3], although both testicular hormones are clearly involved. It has also been demonstrated in male rhesus monkeys that inhibin is the main feedback regulator of the secretion of FSH through actions directly on the pituitary gland [25–30]. In male monkeys, inhibin B is the predominant form of circulating inhibin [31–34], and it may be the physiologically relevant form of inhibin in terms of regulating FSH secretion. In rams, the principal form of inhibin in the circulation has not been determined, and which form of inhibin is responsible for the negative feedback regulation of FSH secretion is not known. Despite the importance of inhibin as a feedback regulator of FSH secretion in both rams and male monkeys, there appears to be a difference between these species, in that testosterone does not seem to be involved in the regulation of FSH secretion in male monkeys [25, 27, 29, 30, 35, 36]. In rams, testosterone does have direct actions that suppress FSH secretion during the breeding season [3] and is capable of synergizing with inhibin in the regulation of FSH secretion during the nonbreeding season [3]. Importantly, the current results demonstrate that these feedback actions of inhibin and testosterone in rams are not influenced by the degree of pituitary stimulation by GnRH.

No effect of treatment in HPD castrated rams with testosterone propionate was observed on the secretion of LH, verifying that testosterone does not act at the level of the pituitary to regulate LH secretion in rams. This corroborates our previous studies in rams, in which we conclusively demonstrated that testosterone does not have pituitary actions to regulate the secretion of LH [1, 3, 7]. Similarly, in male rhesus monkeys, testosterone does not act directly at the pituitary to regulate the secretion of LH [25, 27, 29, 30, 35–37]. In contrast to testosterone, inhibin may have direct pituitary actions to affect LH secretion in rams, because the amplitude of LH pulses was reduced following treatment with rh-inhibin at the slow, but not at the fast, GnRH pulse frequency. Although a statistically significant reduction in the plasma concentrations of LH was observed at both GnRH pulse frequencies, this parameter was highly variable in both control and treated animals. Thus, it cannot be used to assess the influence of the degree of GnRH stimulation on the pituitary actions of testicular hormones in the HPD model. Meaningful assessment can only be made from the amplitude of LH pulses, which are indicative of direct responses to the GnRH stimulus. Therefore, the suppressive effect of rh-inhibin on LH secretion occurred only at the slow GnRH pulse frequency. The finding that rh-inhibin suppressed LH secretion differs from the findings in studies of male rhesus monkeys, in which it has been repeatedly shown that inhibin does not affect LH secretion [25, 27–30, 38], and from those in our previous studies, in which secretion of LH was not affected by administration of rh-inhibin in HPD castrated rams infused with GnRH every 2 h [1, 3] and in castrated rams with an intact hypothalamopituitary unit [1, 2]. Nonetheless, the frequencies of GnRH pulses in the rams of these previous studies were at least double the slow frequency of pulses in this experiment, in which the amplitude of LH pulses was decreased following treatment with rh-inhibin. Clearly, when the GnRH drive to the pituitary is moderate to high, inhibin does not suppress the secretion of LH in rams, but it also appears that under some conditions, when the GnRH pulse frequency is slow, inhibin may suppress the secretion of LH. There have been other reports in which various inhibin preparations have been shown to suppress the secretion of LH in vivo in rams [39, 40], ewes [41–43], and male rats [44–46], but this often occurred only when high doses of the inhibin preparations were administered, the results were frequently variable, the suppressive effects of inhibin on LH secretion were usually less than those on FSH secretion, and inhibin was regularly concluded to be a major feedback regulator of FSH, but not of LH, secretion. The mechanisms and physiological importance of the actions of inhibin at the pituitary to affect LH secretion in rams are unknown. Overwhelming evidence exists that the predominant feedback regulator of LH secretion in rams is testosterone, acting either directly or, via conversion to its primary metabolites, at the level of the hypothalamus to affect the synthesis and/or secretion of GnRH [47]. Therefore, the role of inhibin in the feedback regulation of LH secretion in rams is most likely of minor physiological importance compared to that of testosterone.

Our data suggest that the mechanisms by which inhibin and testosterone regulate the secretion of FSH in rams are unlikely to depend on events that are mediated via GnRH receptors, but the mechanisms of action for these testicular hormones at the pituitary are unknown. Studies in male rats have shown that testosterone can influence the levels of mRNA for the gonadotropin subunits [48], but to our knowledge, similar experiments have not been reported for rams. Inhibin has been shown to reduce the mRNA for the FSHß subunit in a number of species, including male rhesus monkeys [29], rats [49–51], and hamsters [52]. Although inhibin receptors and inhibin-activated signal transduction pathways have not been determined, two classes of binding sites for inhibin on ovine pituitary were recently identified [53], and an inhibin-binding protein was purified from bovine pituitaries [54]. Also, it was shown in cell culture systems that the type III transforming growth factor-ß receptor betaglycan can function as an inhibin coreceptor, with the activin receptor ActRII facilitating antagonism of activin [55]. Finally, we have suggested that inhibin may suppress the levels of mRNA for the FSHß subunit through mechanisms that do not involve transcription of the gene, possibly by influencing factors that interact with the 3'-untranslated region of the FSHß gene to destabilize the FSHß mRNA [56, 57]. Nonetheless, this hypothesis has not been tested.

The frequency of GnRH pulses affected the plasma concentrations of FSH and LH and the amplitude of LH pulses in a manner consistent with findings from other studies using HPD sheep [58–60]. Although the effect of changing the frequency of GnRH pulses on the secretion of the gonadotropins has been investigated in HPD testicular-intact rams [60], this is the first study, to our knowledge, in which these relationships have been examined in HPD castrated rams treated with combinations of inhibin and testosterone. Our results demonstrate that the relationships between GnRH pulse frequency and the secretion of FSH and LH in rams are stable irrespective of the feedback actions of testicular hormones, and that they are similar to the relationships observed in ewes. For instance, in HPD ovariectomized ewes, decreases in GnRH pulse frequency led to increases in LH pulse amplitude, decreases in plasma LH baseline, and increases in plasma concentrations of FSH [58] that are identical to the responses found in the current experiment with HPD castrated rams. These relationships illustrate the differential control of the secretion of gonadotropins by GnRH in rams. It has been hypothesized that LH is dependent on GnRH for both its synthesis and its secretion, whereas GnRH is only required for the synthesis of FSH (because its secretion is passive) [61, 62]. Indeed, substantial evidence exists that the secretion of FSH has a GnRH-independent component [63]. The relative importance of GnRH for the synthesis and secretion of the gonadotropins in rams has been illustrated by studies in which passive immunization against GnRH [64], administration of a GnRH antagonist [65], and treatment with a GnRH agonist [66–68] resulted in an immediate blockade of LH pulses but no acute change in plasma concentrations of FSH. In rams, a high degree of concordance is observed between pulses of GnRH in hypophysial portal plasma and LH in peripheral plasma [5–7], but FSH is not secreted in pulses that are temporally related to GnRH pulses [47]. Furthermore, pulses of LH in HPD rams can be established in response to pulsatile administration of GnRH, but the secretion of FSH is nonpulsatile [60]. It has been proposed based on research in females that the GnRH pulse frequency alters the releasable pool of LH in the pituitary gland such that, during fast frequencies, the pool is small and the LH pulse amplitude is correspondingly small, with the opposite occurring during slow frequencies [61]. The inverse relationship between the frequency of GnRH pulses and the amplitude of LH pulses observed in the HPD castrated rams in this experiment suggests that this proposal also applies to rams.

In summary, the results of this study do not support the hypotheses that were tested, indicating that stimulation of the pituitary gland by GnRH in rams does not influence the actions of inhibin or testosterone to regulate FSH secretion or the actions of testosterone to affect LH secretion. The data from this experiment confirmed previous findings that the negative feedback regulation of FSH secretion in rams involves the actions of both inhibin and testosterone at the level of the pituitary gland, although inhibin is more potent than testosterone as a negative feedback regulator of FSH secretion. In contrast to the actions of inhibin on FSH secretion in rams, secretion of LH can be inhibited by inhibin when the GnRH drive to the pituitary gland is reduced. Nonetheless, the physiological importance of this action is yet to be elucidated, and testosterone is clearly the predominant feedback regulator of LH secretion in rams. Furthermore, we provide evidence to show that testosterone does not act at the level of the pituitary gland to regulate LH secretion in rams.

ACKNOWLEDGMENTS

We thank Bruce Doughton, Alison Skinner, Tony Pisano, Michelle Serapiglia, and Maree Purdon for technical assistance. We thank Dr. Anne Turner for her assistance with the statistical analysis and for constructive criticisms on the manuscript.

FOOTNOTES

First decision: 9 June 2000.

1 Supported by the National Health and Medical Research Council of Australia. Back

2 Correspondence: Alan J. Tilbrook, Department of Physiology, P.O. Box 13F, Monash University, Victoria, 3800, Australia. FAX: 61 3 9905 2547;alan.tilbrook{at}med.monash.edu.au Back

Accepted: September 11, 2000.

Received: May 12, 2000.

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